Cyanobacteria carrying ansmt-lux transcriptional fusion as biosensors for the detection of heavy metal cations

The metal-responsivesmt operator/promoter region ofSynechococcus PCC7942 was fused to theluxCDABE genes ofVibrio fischeri. Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria.Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl₂ concentrations within a range of 0.5–4 μM as demonstrated by induction of luminescence. Trace levels of CuSO₄, and CdCl₂ were also detected.     

16S rRNA-Targeted identification of cyanobacterial genera using oligonucleotide-probes immobilized on bacterial magnetic particles

16S rRNA-targeted identification of cyanobacterial genera, Anabaena,Microcystis, Nostoc, Oscillatoria, Synechococcus wasdeveloped using bacterial magnetic particles (BMPs). 16S rRNA-targetedcapture probes designed from the genus specific region of the 16S rRNAsequence were immobilized on BMPs. Identification of cyanobacteria wasperformed by a sandwich hybridization using the capture probes – BMPconjugates and a digoxigenin (DIG)-labeled detector probe complementaryto the highly conserved 16S rRNA sequence for cyanobacteria. Theluminescence intensity of the probe/target-BMP hybrids was measured afterreaction with alkaline phosphatase conjugated anti-DIG antibody. Fivespecies of cyanobacteria from five different genera were successfullydiscriminated using this magnetic capture system.     

两株聚球藻伴生细菌分离纯化与促生功能鉴定

【背景】蓝藻周围存在伴生细菌,伴生细菌与蓝藻具有复杂的作用关系。【目的】研究淡水聚球藻伴生细菌对聚球藻生长的影响。【方法】采用高通量测序分析聚球藻伴生细菌多样性;平板划线法纯化聚球藻伴生细菌,通过形态观察结合16S rRNA基因序列同源性比对,对其种属关系进行确定;通过聚球藻和不同浓度伴生细菌共培养测定其叶绿素a浓度,分析伴生细菌对聚球藻生长的影响;采用种子发芽试验验证伴生细菌促生功能。【结果】淡水聚球藻伴生细菌优势菌属为产卟啉杆菌属(Porphyrobacter)、根瘤菌属(Rhizobium)、水单胞菌属(Aquimonas)和中慢生根瘤菌属(Mesorhizobium),从聚球藻分离获得了两株伴生细菌JQ1和JQ2,基于16S rRNA基因序列鉴定其分别属于Rhizobium和Peribacillus,通过在聚球藻与不同浓度伴生细菌共培养及水稻发芽试验验证,证明伴生细菌JQ1和JQ2在菌藻比例分别为5:1和15:1时具有促生作用,都对增强秧苗素质和根系发育有一定影响但JQ2与JQ1相比能显著提高水稻种子的发芽率。【结论】淡水聚球藻伴生细菌JQ1和JQ2在适宜的浓度均可显著促进聚球...     

HIP1 propagates in cyanobacterial DNA via nucleotide substitutions but promotes excision at similar frequencies in Escherichia coli and Synechococcus PCC 7942

The sequence 5'-GCGATCGC- 3', designated HIP1, for highly iterated palindrome, was first identified at the borders of a gene-deletion event and subsequently shown to constitute up to 2.5% of the DNA in some cyanobacteria. It is now reported that HIP1 is polyphyletic, occurring in several distinct cyanobacterial lineages and not defining a clade. HIP1 does not introduce gaps into sequence alignments. It aligns with partial HIP1 sites in related sequences showing that it propagates by nucleotide substitutions rather than insertion. Constructs have been created to determine the frequencies at which deletion events occur between palindromes located within the selectable marker neo Deletion between HIP1 sites was more frequent in Synechococcus PCC 7942 than deletion between control palindromes, 5'-CCGATCGG-3', designated PAL0. However, this is not due to a recombinase that recognises HIP1 and is peculiar to cyanobacteria because similar deletion frequencies were detected in Escherichia coli . Furthermore, the frequency of deletion of DNA flanked asymmetrically by one HIP1 site and one PAL0 site was less than the frequency of deletion of DNA flanked symmetrically by identical copies of either palindrome. This is consistent with deletion by copy-choice.     

Fine-scale distribution patterns of Synechococcus ecological diversity in microbial mats of Mushroom Spring, Yellowstone National Park

Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-μm vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O₂, pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O₂ concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Functional characterization of the Aspergillus nidulans glucosylceramide pathway reveals that LCB Δ8‐desaturation and C9‐methylation are relevant to filamentous growth,lipid raft localization and Psd1 defensin activity

C8‐desaturated and C9‐methylated glucosylceramide (GlcCer) is a fungal‐specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8‐desaturase (SdeA), sphingolipid C9‐methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8‐saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild‐type cells showed a similar 9,Me‐GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me‐GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.     

COMPARATIVE MOLECULAR EVOLUTION OF NEWLY DISCOVERED PICOCYANOBACTERIAL STRAINS REVEALS A PHYLOGENETICALLY INFORMATIVE VARIABLE REGION OF β‐PHYCOERYTHRIN1

The genetic diversity and phylogenetic position of 10 strains of picocyanobacteria from the Arabian Sea were examined using partial sequences from three loci: 16S rDNA, RNA polymerase rpoC1, and two elements of the phycoerythrin (PE) locus, cpeA and cpeB which encode for the α and β subunit of PE. Nine of the strains showed nearly identical spectral phenotypes based on the in vivo excitation spectrum for PE fluorescence emission and appear to be strains synthesizing a phycourobilin (PUB)–lacking PE. These strains include one, Synechococcus sp. G2.1, already known to be closely related to filamentous cyanobacteria and not to the commonly studied 5.1 subcluster of marine Synechococcus. The 10th strain was a PE‐lacking strain that was of interest because it was isolated from open‐ocean conditions where picocyanobacteria with this phenotype are relatively uncommon. Phylogenetic analysis of the concatenated 16S rDNA and rpoC1 data sets showed that none of the previously described strains were members of the 5.1 subcluster of marine Synechococcus, nor were they closely related to strain G2.1. Instead, they form a well‐supported and previously undescribed clade of cyanobacteria that is sister to Cyanobium. Thus, these strains represent the first PE‐containing Cyanobium from oceanic waters, and the lineage they define includes a strain with a PE‐lacking phenotype from the same environment. Analysis of the PE sequence data showed the PE apoprotein has evolved independently in the G2.1 lineage and the Cyanobium‐like lineage represented by the study strains. It also revealed a hypervariable region of the β‐subunit not described previously; variation in this region shows a pattern among a wide range of PE‐containing organisms congruent with the phylogenetic relationships inferred from other genes. This suggests that the PUB‐lacking spectral phenotype is more likely to have evolved in distantly related phylogenetic lineages by either divergent or convergent evolution than by lateral gene transfer. Both the conserved PE gene sequences and the inferred amino acid sequences for the hypervariable region show considerable divergence among Prochlorococcus PEs, red algal PEs, PUB‐containing PEs from the marine Synechococcus 5.1 subcluster, PEs from the Cyanobium‐like strains, and PEs from other cyanobacteria (including strain G2.1). Thus, it appears that the hypervariable region of the PE gene can be used as a taxon‐specific marker.     

Synechococcus: 3 billion years of global dominance

Cyanobacteria are among the most important primary producers on the Earth. However, the evolutionary forces driving cyanobacterial species diversity remain largely enigmatic due to both their distinction from macro‐organisms and an undersampling of sequenced genomes. Thus, we present a new genome of a Synechococcus‐like cyanobacterium from a novel evolutionary lineage. Further, we analyse all existing 16S rRNA sequences and genomes of Synechococcus‐like cyanobacteria. Chronograms showed extremely polyphyletic relationships in Synechococcus, which has not been observed in any other cyanobacteria. Moreover, most Synechococcus lineages bifurcated after the Great Oxidation Event, including the most abundant marine picoplankton lineage. Quantification of horizontal gene transfer among 70 cyanobacterial genomes revealed significant differences among studied genomes. Horizontal gene transfer levels were not correlated with ecology, genome size or phenotype, but were correlated with the age of divergence. All findings were synthetized into a novel model of cyanobacterial evolution, characterized by serial convergence of the features, that is multicellularity and ecology.     

Characterization of Trichodesmium spp. by Genetic Techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus Trichodesmium. The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of Katagnymene. Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

Characterization of the single psbA gene of Prochlorococcus marinus CCMP 1375 (Prochlorophyta)

DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P. marinus CCMP 1375 was analyzed. The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence. P. marinus contains only a single psbA gene. Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do. Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P. Marinus separately from Prochlorothrix hollandica among the cyanobacteria.     

Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium

Background  

Many bacteria swim by rotating helical flagellar filaments [1]. Waterbury et al. [15] discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces [2,7]. The hypothesis that Synechococcus might swim using traveling surface waves [6,13] prompted this investigation.     

Codon usage patterns and adaptive evolution of marine unicellular cyanobacteria Synechococcus and Prochlorococcus

Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d_N/d_S values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence.     

Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated<Emphasis Type="Italic"> gus</Emphasis> transgenes in Arabidopsis

The transgene locus KH15, which is highly susceptible to silencing in Arabidopsis thaliana, contains two inversely repeated beta-glucuronidase (gus) genes separated by a palindromic sequence and has a low GUS activity, was found to be heavily methylated in the gus coding sequence and in the center of the inverted repeat. The locus KHsb67, which is less prone to silencing, was found to be less densely methylated in the non-repetitive region that separates the inversely repeated gus genes. After the removal of one of the gus genes by Cre-mediated recombination, methylation in both loci decreased or was totally lost. Despite the presence of a 732-bp palindromic sequence in the deletion line derived from KH15, this sequence was not methylated. Whereas the KH15 locus triggers methylation of homologous gus genes when placed in trans to them, the deletion derivative did not, suggesting that the capacity for cross-talk was severely affected by disruption of the palindromic arrangement. This result suggests that the transcribed palindromic sequences are required to maintain the methylation of both symmetrically and non-symmetrically arranged cytosines.     

Functional Differences Between Cyanobacterial DnaK1 Chaperones from the Halophyte Aphanothece halophytica and the Freshwater Species Synechococcus elongatus Expressed in Escherichia coli

DnaK chaperones participate in essential cellular processes including the assistance of the folding, structural maintenance, trafficking, and degradation of proteins, the control of stress responses, and so on. In contrast to the situation found in most other bacterial groups, the cyanobacteria contain multiple dnaK homolog genes whose cellular roles remain ambiguous. We compared in this work the in vivo chaperone capabilities of the DnaK1 members from the halophyte Aphanothece halophytica and the freshwater species Synechococcus elongatus. The corresponding dnaK1 genes were expressed in Escherichia coli, and the abilities of the encoded chaperones to provide for both general and specific functions conducted by E. coli DnaK were analyzed. Synechococcus DnaK1 was far more effective than A. halophytica DnaK1 in replacing E. coli DnaK in all activities tested in vivo, including changes in cell morphology and downregulation of the heat shock response, prevention of the aggregation of misfolded proteins, and restoration of thermotolerance to dnaK-deficient mutants. Thus, regardless of an extensive sequence similarity and comparable in vitro chaperone capabilities, the two cyanobacterial DnaK1 chaperones functionally differed under in vivo conditions. The overall results reinforce the notion that A. halophytica DnaK1 and Synechococcus DnaK1 evolved different substrate specificity since they separated from a common ancestor.     

Effects of photosynthetic inhibitors and light-dark regimes on the replication of cyanophage SM-2

The effects of photosynthetic inhibitors and light-dark regimes on the replication of cyanophage SM-2 in its host cyanobacteria (Synechococcus elongatus UTEX 563 and Microcystis aeruginosa NRC-1, Synechococcus NRC-1 UTEX 1937) have been investigated. Photoassimilation of CO₂ by infected cells was enhanced and remained elevated until late in the infection cycle. Photosynthetic inhibitors and the removal of light suppressed viral replication. SM-2, like other cyanophage of unicellular cyanobacteria, is highly dependent on host photosynthetic metabolism for the energy required in replication.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CCCP carbonyl-cyanide m-chlorophenyl hydrazone - MMH Modified Modified Hughes Medium